Decoding the "Procainamide" paper


1.  What is a tumor suppressor?  What do they do?  How do they do it?
A tumor suppressor gene is a gene that reduces the probability that a cell, in a multicellular organism, will turn into a tumor cell. A mutation or deletion of this gene will increase the probability of the formation of a tumor.
    Tumor suppressor genes, or the proteins for which they code, have a dampening or repressive effect on the regulation of the cell cycle (cell division) and/or promote apoptosis (programmed cell death). Tumor suppressor proteins perform the following functions: (1) inhibit cell division by repressing certain genes that are essential for continuing the cell cycle, (2) couple the cell cycle to DNA damage, so that the cell will not divide (if damage is repaired the cell cycle can continue), and (3) initiate apoptosis if damage cannot be repaired.
    Two examples of tumor suppressor proteins are the pRb protein and the p53 gene. The normal Rb protein prevents mitosis. The Rb protein prevents the cell form entering the S phase of the cell cycle by binding to a transcription factor called E2F. This prevents E2F from binding to the promoters of such proto-oncogenes as c-myc and c-fos, which are needed for mitosis. The p53 protein prevents a cell from completing the cell cycle if its DNA is damaged. When the damage is minor, p53 stops the cell cycle until the damage is repaired. When the damage is major, p53 triggers the cell to commit suicide by apoptosis. P53 is a key player in protecting people against cancer.

http://users.rcn.com/jkimball.ma.ultranet/ BiologyPages/T/TumorSuppressorGenes.html

http://en.wikipedia.org/wiki/Tumor_suppressor_gene

2.  What is myelodysplastic syndrome?
Myelodysplastic syndromes used to be called preleukemia because some people with a myelodysplastic syndrome develop leukemia.  It is no longer referred to preleukemia because so few with the syndrome develop leukemia. However if leukemia does develop, it is more difficult to treat then primary leukemia (which is when a patient has had no other bone marrow disease).
Myelodysplastic syndromes are a group of diseases that affect blood cells via the bone marrow.  The bone marrow in Myelodysplastic syndrome is generally more active then healthy bone marrow, and yet the amount of blood cells in the body is reduced.  Another feature of this syndrome is a change in the way the bone marrow and red blood cells look. 
People who are most likely to develop this syndrome are older patients, the average age is 65 to 75.  Some of the symptoms associated with myelodysplastic syndrome are anemia including shortness of breath and chilled sensation, neutropenia an increased susceptibility to infection, and thrombocytopenia, which is susceptibility to, increased bleeding.

3.  What is neutropenia?  Thrombocytopenia?
Neutropenia:
    Neutrophils are the body’s first line of defense and work by going to the site of infections, damage, or inflammation to join "the battle" and by causing phagocytosis of particles such as bacteria.  They normally increase when there is an infection as part of a persons "immune defense system".  When a person’s immune system becomes compromised and is at increased risk for infection doctors look at more then the WBC’s (white blood cells). They actually look at the differential on the CBC and calculate the ANC (absolute neutrophil count). By doing this they are able to look at the precursors of the WBC’s. These are the "baby WBC’s" or immature blood cells. These are found in the patient’s bone marrow. This number gives a more accurate measurement of a persons risk for infection. An ANC of less than 2000 is diagnosed as neutropenia.

Thrombocytopenia:
    Thrombocytopenia is any disorder in which there are not enough platelets. Platelets are cells in the blood that help blood to clot. This condition is sometimes associated with abnormal bleeding.

4.  How does the DNeasy tissue kit work for genomic DNA isolation?
Several methods of isolation and purification of DNA are now available in scientific communities around the world.  DNeasy Tissue Kit is one of the major technological tools used to quickly isolate DNA material from a variety of sample sources.  An advanced silica-gel membrane technology in the kit enables a quick and efficient purification of DNA material without additional organic extraction or precipitation.  The DNeasy tissue kit works by executing a simple procedure where sample cells are first lysed with proteinase K.  Under optimal conditions provided by certain buffers, DNA is selectively bound to the DNeasy membrane as contaminants pass trough during a short centrifugation.  Contaminants and enzymes inhibitors are then washed away to leave an isolated genomic DNA.

www.qiagen.com

5.  How is HPLC -MS used to quantitate methylated dC?


HPLC-MS
Check out this power point presentation by Knut Reinert.  There is some very good information about proteomics and the cutting edge technology that is being used at the Berlin Center for Bioinformatics.  


6.  What is bisulfite genomic sequence analysis?

Bisulfite reacts with unmethylated Cytdidine bases, after forming a sulfoxide adduct, the cytidine deaminates.  The sulfoxide is removed with sodium hydroxide and the resulting product is Uracil.  When the DNA is sequenced, a T shows up where a G should be.  The methylated C bases will not be deaminated to Uracil, so a G will be where a G was in the original sequence.

3' ATCCGTA 5'
Sequence:
5' TAGGCAT3'

Reaction with bisulfite:
3' ATUUGTA5'
Sequence:
5" TAAACAT3'

If the original sequence has a methylated C, then it will not be converted to U.

3' ATMe-CCGTA 5'
Sequence after reaction with Bisulfite:
5' TAGACAT3'


http://docs.appliedbiosystems.com/pebiodocs/00113199.pdf

7.  What is PCR?  How does it work?
     PCR stands for Polymerase Chain Reaction, a process discovered in 1983, that is used to amplify specific DNA sequences.  This process is so sensitive that even a single DNA molecule can be detected and its target sequence amplified about a million-fold. There are three essential steps to the PCR procedure.  First, the DNA double helix is heated to separate the two DNA strands.  Second, each separated strand is annealed to a synthetic oligonucleotide DNA primer on the end of the region to be sequenced (annealed is just another term for the joining, or base pairing, of the DNA).   Thus, the synthetic primers “flank” the region of DNA to be amplified.  It should be noted that these synthetic primers are present in excess as the mixture is cooled so that the likelihood of the primer binding to the single-stranded DNA is greater than the likelihood of the two separated DNA strands rejoining. The third step of the procedure is the synthesis of new DNA, beginning at the primers.  This synthesis is accomplished by a heat-stable DNA polymerase (usually TaqI polymerase).  After the third step is completed, the whole cycle of heating, annealing, and synthesizing DNA is repeated (usually for about 25 to 30 cycles).  The reason for using the heat-stable DNA polymerase is that the enzyme does not denature after each heating process, so it remains active after each time the mixture is heated and its supply does not have to be replenished after each cycle.
    PCR is an extremely valuable tool that is used in several areas of science, such as human genome sequencing and forensic medicine.   In general medicine, PCR can be used to detect the presence of even minute quantities of virus, thus providing early detection of viral infections before any symptoms are present.  PCR is also used for prenatal diagnosis of many different genetic diseases.  Archaeologists employ the PCR method to amplify the DNA of mummified human remains as well as the DNA of extinct animals.  In its twenty years of existence, the PCR method of DNA amplification has been an incredibly useful procedure and will continue to aid in the advancement of many areas of science.

References
Nelson, David L. and Michael M. Cox.  Lehninger Principles of Biochemistry.  4th ed.
    New York: W.H. Freeman and Company, 2005.


8.  What is a Southern blot?  What is it used for?  How does it work?
    Southern blot is a technique developed by Edward Southern in which DNA fragments produced by a restriction enzyme digestion are separated by electrophoresis and transferred by capillary action to a nylon or nitrocellulose membrane.  Specific DNA fragments can be identified by hybridization to a labeled nucleic acid probe.          
     To make a southern blot, DNA is cut into fragments with one or more restriction enzymes and the fragments are separated by gel electrophoresis. The DNA in the gel is usually stained and photographed or scanned to reveal the number and molecular weights of the restriction fragments.  The DNA in the gel is then denatured to form single-stranded fragments by treating the gel with an alkaline solution. The gel is then overlaid with a membrane of DNA-binding material, usually nitrocellulose or a nylon derivative. To transfer the fragments, the membrane and gel are placed are placed on a wick (often a sponge) in contact with a buffer solution.  The buffer flows through the wick, gel and membrane by capillary action. As the buffer solution flows, the DNA fragments move out of the gel and become immobilized on the membrane.
      The DNA fragments on the membrane are hybridized with a labeled, single-stranded DNA probe. Only DNA fragments complementary to the probes nucleotide sequence will form double-stranded hybrids.  Excess probe is washed away and the hybridized fragments are visualized on a piece of film.    
      The southern blot method can be used to identify which clones in a library contain a given DNA sequence (such as ribosomal DNA). Southern blots are  also be used to determine whether a clone contains all or only part of the gene and to ascertain the overall size and sequence of a gene or DNA sequence of interest. Some other uses include detection of rearranged, deleted and duplications of genes associated with human genetic disorders and cancers. 

http://www.accessexcellence.org/RC/VL/GG/southBlotg.html

9.  What is an alphoid satellite sequence?
     This is a sequence of DNA that is highly repeated.  This 171 bp pair sequence is found on the centromeres of chromosomes.  While the sequence is conserved for each chromosome, the number of copies of it are highly variable.  TThere can be 1,000 to 1,000, 000 repeats of this sequence.  It is 3-5% of the DNA in the Y chromosome.   A related type of tandem DNA repeats are microsatellites.  The number of repeats  in microsattelites an be used to establish genetic relationships between ancestors and progeny.  Check out the link to the Thomas Jefferson heirs.

Back to the Main Biochemistry page at CSU, Stanislaus
last updated: March 7, 2006